Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2'dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

Understanding and controlling the metal-directed assembly of terpyridine-functionalized coiled-coil peptides.

Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.

Unmasking the pathological and therapeutic potential of histone deacetylases for liver cancer.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, currently ranking as one of the highest neoplastic-related mortalities in the world. Due to the difficulty in early diagnosis and lack of effective treatment options, the 5-year survival rate of HCC remains extremely low. Histone deacetylation is one of the most important epigenetic mechanisms, regulating cellular events such as differentiation, proliferation and cell cycle. Histone deacetylases (HDACs), the chief mediators of this epigenetic mechanism, are often aberrantly expressed in various tumours including HCC. Areas covered: This review focuses on the most up-to-date findings of HDACs and their associated molecular mechanisms in HCC onset and progression. In addition, a potential network between HDACs and non-coding RNAs including microRNAs and long noncoding RNAs underlying hepatocarcinogenesis is considered. Expert opinion: Unmasking the role of HDACs and their association with HCC pathogenesis could have implications for future personalized therapeutic and diagnostic targeting.

Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy.

There is increasing evidence that the stability, structure, dynamics, and function of many proteins differ in cells versus in vitro. The determination of protein structure and dynamics within the native cellular environment may lead to better understanding of protein behavior. Electron spin resonance (ESR) has emerged as a technique that can report on protein structure and dynamics within cells. Nitroxide based spin labels are capable of reporting on protein dynamics, structure, and backbone flexibility but are limited due to nitroxide reduction occurring in cells. In order to overcome this limitation, we used the oxidizing agent potassium ferricyanide (K3Fe(CN)6) as well as the cleavage resistant spin label 3-malemido-PROXYL (5-MSL). Furthermore, we hypothesized that injection concentration is an important parameter regarding nitroxide reduction kinetics. By increasing the injection concentration of doubly 5-MSL labeled protein into Xenopus laevis oocytes, we found an increased nitroxide lifetime. Our work demonstrates unprecedented incubation times of 3-h in-cell and 5-h in-cytosol for double electron-electron resonance (DEER) experiments using nitroxide spin labels. This allows for more meaningful measurements of larger protein systems which may require longer incubation times for equilibration in the cellular milieu. Even longer incubation times are possible by combining our approach with more shielded nitroxides and Q-band.

Antiviral activity of bone morphogenetic proteins and activins.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Improved Early Postoperative Range of Motion in Total Knee Arthroplasty Using Tranexamic Acid: A Retrospective Analysis.

The use of tranexamic acid (TXA) in total knee arthroplasty (TKA) has become common practice. Recent literature has demonstrated a reduction in postoperative knee swelling and drain output while using TXA. Our purpose is to analyze the range of motion (ROM) following TKA in patients who received TXA compared with a control group. We hypothesize that patients treated with TXA will have improved early postoperative ROM when compared with controls. A retrospective chart review was performed for patients who underwent TKA from 2010 to 2012 performed by a single orthopaedic surgeon. Patients were stratified into three cohorts by route of TXA administration including intravenous (IV), topical, and a control group. Dependent variables analyzed included extension, flexion, and total arc ROM on each postoperative day (POD), average ROM across all three postoperative days, as well as pre-to-postoperative differences in ROM. Demographic data were recorded for each patient. A total of 174 patients were included for analysis, 75 controls and 99 receiving TXA. A significant difference was found between the treatment groups and the control for all variables (for each, p ≤ 0.002). There were no significant differences in ROM between the IV and topical TXA treatment groups (for each, p ≥ 0.558). A multivariate analysis demonstrated no significant difference between the groups in complication rate or demographic variables. The use of TXA may improve early postoperative ROM following TKA.

The therapeutic properties of resminostat for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with increases in new cases being reported annually. Histopathologists have identified hepatic steatosis as a characteristic of a broad range of chronic liver diseases that are associated with the onset and development of HCC. In this context, epigenetic modifications may serve as precancerous factors predisposing normal cells to the initiation of carcinogenesis. This study demonstrated that hepatic tumorigenesis and differentiated adipocytes may modulate both global histone deacetylase (HDAC) expression and specific class I HDAC genes in the tumour microenvironment. The novel class I HDAC inhibitor Resminostat was shown to reduce the proliferation of HCC cells along with its specificity in targeting class I HDACs and oncogenes. The combined effect of Resminostat with several pharmaceutical agents such as Sorafenib, Cisplatin and Doxorubicin was also demonstrated. The inhibition of heat shock protein 90 (HSP90) has been demonstrated as a potential therapeutic option for HCC. In line with this, the specific HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was selected and it was found that the combination of Resminostat and 17-AAG may provide a "smart" clinical strategy for HCC patients by targeting cellular communication within the tumour microenvironment. This study provides an insight into the use of Resminostat as an epigenetic based therapeutic for HCC along with other pharmaceutical options, in particular by targeting the cell-to-cell communication that occurs between hepatoma and adipocytes.

ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1.

Nitroxide- and Cu2+-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important α-helix of the human glutathione S-transferase A1. Distance measurements on various spin-labeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu2+-based ESR distance measurements. Cu2+-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.

The Cu2+-nitrilotriacetic acid complex improves loading of α-helical double histidine site for precise distance measurements by pulsed ESR.

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand ß-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu2+-NTA.

Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

Nucleotide-Independent Copper(II)-Based Distance Measurements in DNA by Pulsed ESR Spectroscopy.

A site-specific Cu2+ binding motif within a DNA duplex for distance measurements by ESR spectroscopy is reported. This motif utilizes a commercially available 2,2'-dipicolylamine (DPA) phosphormadite easily incorporated into any DNA oligonucleotide during initial DNA synthesis. The method only requires the simple post-synthetic addition of Cu2+ without the need for further chemical modification. Notably, the label is positioned within the DNA duplex, as opposed to outside the helical perimeter, for an accurate measurement of duplex distance. A distance of 2.7 nm was measured on a doubly Cu2+ -labeled DNA sequence, which is in exact agreement with the expected distance from both DNA modeling and molecular dynamic simulations. This result suggests that with this labeling strategy the ESR measured distance directly reports on backbone DNA distance, without the need for further modeling. Furthermore, the labeling strategy is structure- and nucleotide-independent.

The Assessment of Blood Loss During Total Knee Arthroplasty When Comparing Intravenous vs Intracapsular Administration of Tranexamic Acid.

BACKGROUND: Administration of tranexamic acid topically and intravenously has demonstrated effectiveness in decreasing blood loss and transfusion rates. METHODS: We randomized 131 patients undergoing primary total knee arthroplasty to receive either intracapsular (69) or intravenous tranexamic acid (62). Postoperative blood loss was calculated using the formula derived by Nadler et al. The number of units transfused was recorded, as well as length of hospital stay. RESULTS: We found no statistically significant difference on calculated blood loss (postoperative day [POD] 1: 624 ± 326 vs 644 ± 292; P = .71, POD 2: 806 ± 368 vs 835 ± 319; P = .64, and POD 3: 1076 ± 419 vs 978 ± 343; P = .55). There was no difference in number of blood transfusions, length of stay, or complications. CONCLUSION: Intracapsular tranexamic acid is not inferior to intravenous tranexamic acid in decreasing blood loss and blood transfusion rate in primary total knee arthroplasty.

Cu(II)-Zn(II) Cross-Modulation in Amyloid-Beta Peptide Binding: An X-ray Absorption Spectroscopy Study.

In this work we analyze at a structural level the mechanism by which Cu(II) and Zn(II) ions compete for binding to the Aß peptides that is involved in the etiology of Alzheimer's disease. We collected X-ray absorption spectroscopy data on samples containing Aß with Cu and Zn at different concentration ratios. We show that the order in which metals are added to the peptide solution matters and that, when Zn is added first, it prevents Cu from binding. On the contrary, when Cu is added first, it does not (completely) prevent Zn binding to Aß peptides. Our analysis suggests that Cu and Zn ions are coordinated to different numbers of histidine residues depending on the [ion]:[peptide] concentration ratio.

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC.

Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using (19)F nuclear magnetic resonance and electron spin resonance spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

Long noncoding RNAs in liver cancer: what we know in 2014.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with an estimated over half a million new cases diagnosed annually. Due to the difficulty in early diagnosis and lack of effective treatment options, HCC is currently ranked as the second highest neoplastic-related mortality in the world, with an extremely low 5-year survival rate of between 6 and 11%. Long noncoding RNAs (lncRNAs), are genes lacking protein coding ability, have recently emerged as pivotal participants in biological processes, often dysregulated in a range of cancers, including HCC. AREAS COVERED: In this review, we highlight the recent findings of lncRNAs in HCC pathogenesis, with particular attention on epigenetic events. In silico analysis was utilized to emphasize intrinsic linkages within the ncRNA families associated with hepatocarcinogenesis. EXPERT OPINION: While our understanding of lncRNAs in the onset and progression of HCC is still in its infancy, there is no doubt that understanding the activities of ncRNAs will certainly secure strong biomarkers and improve treatment options for HCC patients.

Greater trochanteric hip pain.

In the patient with lateral hip pain, there is a broad differential diagnosis, making appropriate evaluation and management challenging. Greater trochanteric pain syndrome is a term used to denote chronic lateral hip pain and encompasses several painful soft tissue diagnoses including coxa saltans, trochanteric bursitis, and gluteus minimus and medius tendon tears. An overview of these common causes is presented through a series of cases that encompass the anatomic associations, classic presentations, diagnostic tests, and management strategies unique to each disorder. By reviewing this information, we hope to provide clinicians with the tools to evaluate greater trochanteric pain syndrome efficiently and effectively.

Stop feeding cancer: pro-inflammatory role of visceral adiposity in liver cancer.

Liver cancer is the fifth most common cancer in the world with an estimated over half a million new cases diagnosed every year. Due to the difficulty in early diagnosis and lack of treatment options, the prevalence of liver cancer continues to climb with a 5-year survival rate of between 6% and 11%. Coinciding with the rise of liver cancer, the prevalence of obesity has rapidly increased over the past two decades. Evidence from epidemiological studies demonstrates a higher risk of hepatocellular carcinoma (HCC) in obese individuals. Obesity is recognised as a low-grade inflammatory disease, this is of particular relevance as inflammation has been proposed as the seventh hallmark of cancer development with abdominal visceral adiposity considered as an important source of pro-inflammatory stimuli. Emerging evidence points towards the direct role of visceral adipose tissue rather than generalised body fat in carcinogenesis. Cytokines such as IL-6 and TNF-α secreted from visceral adipose tissue have been demonstrated to induce a chronic inflammatory condition predisposing the liver to a protumourigenic milieu. This review focuses on excess visceral adiposity rather than simple obesity; particularly adipokines and their implications for chronic inflammation, lipid accumulation, insulin resistance, Endoplasmic Reticulum (ER) stress and angiogenesis. Evidence of molecular signalling pathways that may give rise to the onset and progression of HCC in this context are depicted. Delineation of the pro-inflammatory role of visceral adiposity in liver cancer and its targeting will provide better rational and therapeutic approaches for HCC prevention and elimination. The concept of a central role for metabolism in cancer is the culmination of an effort that began with one of the 20th century's leading biochemists and Nobel laureate of 1931, Otto Warburg.